Methods and means for increasing the tolerance of plants to stress conditions

ABSTRACT

Methods and means are provided to increase the tolerance of plants to abiotic stress or adverse growing conditions, including drought, high light intensities, high temperatures, nutrient limitations and the like by reducing the activity of endogenous PARG proteins in plants.

FIELD OF THE INVENTION

The present invention relates to the use of poly (ADP-ribose)glycohydrolases in plants to increase the tolerance of plants to adversegrowing conditions, including drought, high light intensities, hightemperatures, nutrient limitations and the like. Methods and means areprovided to produce plants that are tolerant to abiotic stressconditions.

BACKGROUND TO THE INVENTION

Frequently, abiotic stress will lead either directly or indirectly todamage of the DNA of the cells of the plants exposed to the adverseconditions. Genomic damage, if left unrepaired, can lead to cell death.Tolerance to stress conditions exhibited by plants is the result of theability of the plant cells exposed to the adverse conditions to reduceand/or repair the damage, and to survive.

Plant cells, like other eukaryotic cells, have evolved an elaborate DNArepair system. The activation of poly(ADP-ribose) polymerase (PARP) byDNA strand breaks is often one of the first cellular responses to DNAdamage. PARP catalyzes the post-translational modification of proteinsby adding successively molecules of ADP-ribose, obtained from theconversion of nicotineamide dinucleotide (NAD), to form multibranchedpolymers containing up to 200 ADP-ribose residues (about 40 residues inplants). The dependence of poly(ADP-ribose) synthesis on DNA strandbreaks, and the presence of PARP in multiprotein complexes furthercontaining key effectors of DNA repair, replication and transcriptionreactions, strongly suggests that this posttranslational modification isinvolved in metabolism of nucleic acids, and DNA repair. There are alsoindications that poly (ADP-ribose) synthesis is involved in regulationof cell cycle and cell death.

Poly (ADP-ribosylation) of proteins is transient in living cells. Thepoly (ADP-ribose) polymers are rapidly turned over, being converted tofree ADP-ribose by the exoglycosidase and endoglycosidase activity ofpoly (ADP-ribose) glycohydrolase (PARG; E.C.3.2.1.143). The mostproximal unit of ADP ribose on the protein acceptor is hydrolyzed by theaction of another enzyme (ADP-ribosyl protein lyase).

In addition to this positive (DNA-repair associated) effect of PARP oncell survival, there is also a negative effect of PARP. The process ofactivating PARP upon DNA damage is associated with a rapid lowering ofNAD+ levels, since each ADP-ribose unit transferred by PARP consumes onemolecule of NAD+. NAD+ depletion in turn results in ATP depletion,because NAD+ resynthesis requires at least (depending on thebiosynthesis pathway) three molecules of ATP per molecule of NAD+.Furthermore, NAD+ depletion block glyceraldehyde-3-phosphatedehydrogenase activity, which is required to resynthesize ATP duringglycolysis. Finally, NAD+ is a key carrier of electrons needed togenerate ATP via electron transport and oxidative phosphorylation.

The physiological consequence of NAD+ and ATP depletion has beenestablished in the context of DNA-damage induced cell death. It has beenshown that the completion of apoptosis is absolutely dependent on thepresence of ATP and that, in the absence of this nucleotide, the type ofcellular demise switches from apoptosis to necrosis. Since the cellularlysis associates with necrosis generates further damage to neighboringcells it is preferable for multicellular organisms to favor apoptoticcell death rather than necrosis.

It is thus very important to consider the delicate balance of positiveand negative effects of the poly (ADP ribosyl)ation on the potential ofa cell to survive DNA damage.

WO 00/04173 describes methods to modulate programmed cell death (PCD) ineukaryotic cells and organisms, particularly plant cells and plants, byintroducing of “PCD modulating chimeric genes” influencing theexpression and/or apparent activity of endogenous poly-(ADP-ribose)polymerase (PARP) genes. Programmed cell death may be inhibited orprovoked. The invention particularly relates to the use of nucleotidesequences encoding proteins with PARP activity for modulating PCD, forenhancing growth rate or for producing stress tolerant cells andorganisms.

PARG encoding genes have been identified in a number of animals such asRattus norvegicus (Accession numbers: NM_(—)031339, NW_(—)043030,AB019366,), Mus musculus (Accession numbers: NT_(—)039598, NM_(—)003631,AF079557), Homo sapiens (Accession numbers: NT_(—)017696; NM_(—)003631,AF005043), Bos taurus (Accession numbers: NM_(—)174138, U78975)Drosophila melanogaster (Accession number: AF079556)

In plants, a poly(ADP-ribose) glycohydrolase has been identified bymap-based cloning of the wild-type gene inactivated in a mutant affectedin clock-controlled transcription of genes in Arabidopsis and inphotoperiod dependent transition from vegetative growth to flowering(tej). The nucleotide sequence of the gene can be obtained fromnucleotide databases under the accession number AF394690 (Panda et al.,2002 Dev. Cell. 3, 51-61).

SUMMARY OF THE INVENTION

The invention provides a method to produce a plant tolerant to stressconditions comprising the steps of providing plant cells with a chimericgene to create transgenic plant cells, wherein the chimeric genecomprises the following operably linked DNA fragments: aplant-expressible promoter; a DNA region, which when transcribed yieldsan ParG inhibitory RNA molecule; and a 3′ end region involved intranscription termination and polyadenylation. A population oftransgenic plant lines is regenerated from the transgenic plant cell;and a stress tolerant plant line is identified within the population oftransgenic plant lines. The ParG inhibitory RNA molecule may comprise anucleotide sequence of at least 20 consecutive nucleotides of thenucleotide sequence of the ParG gene present in the plant cell (theendogenous ParG gene). The ParG inhibitory RNA molecule may alsocomprise a nucleotide sequence of at least 20 consecutive nucleotides ofthe complement of the nucleotide sequence of the ParG gene present inthe plant cell (the endogenous ParG gene). In yet another embodiment,the parG inhibitory RNA may comprise a sense region comprising anucleotide sequence of at least 20 consecutive nucleotides of thenucleotide sequence of the ParG gene present in the plant cell and anantisense region comprising a nucleotide sequence of at least 20consecutive nucleotides of the complement of the nucleotide sequence ofthe ParG gene present in the plant cell, wherein the sense and antisenseregion are capable of forming a double stranded RNA region comprisingsaid at least 20 consecutive nucleotides. The chimeric gene may furthercomprise a DNA region encoding a self-splicing ribozyme between said DNAregion coding for parG inhibitory RNA molecule and the 3′ end region.Stress conditions may be selected from heat, drought, nutrientdepletion, oxidative stress or high light conditions.

In another embodiment of the invention, a method is provided to producea plant tolerant to stress conditions comprising the steps of: isolatinga DNA fragment of at least 100 bp comprising a part of the parG encodinggene of the plant of interest; producing a chimeric gene by operablylinking a plant expressible promoter region to the isolated DNA fragmentcomprising part of the parG encoding gene of the plant in directorientation compared to the promoter region; and to the isolated DNAfragment comprising part of the parG encoding gene of said plant ininverted orientation compared to the promoter region, and a 3′ endregion involved in transcription termination and polyadenylation. Thesechimeric genes are then provided to plant cells to create transgenicplant cells. A population of transgenic plant lines is regenerated fromthe transgenic plant cells; and a stress tolerant plant line isidentified within the population of transgenic plant lines. Theinvention also relates to stress tolerant plant cells and plantsobtained by this process.

In yet another embodiment of the invention, a method is provided toproduce a plant tolerant to stress conditions comprising the steps ofproviding plant cells with a chimeric gene to create transgenic plantcells, comprising a DNA region, which when transcribed yields an ParGinhibitory RNA molecule, whereby the DNA region comprises a nucleotidesequence of at least 21 to 100 nucleotides of a nucleotide sequenceencoding a protein comprising the amino acid sequence of SEQ ID No 1, 2or 16 or at least 21 to 100 nucleotides of a nucleotide sequence of SEQID 3, 4, 15 or 23 operably linked to a plant-expressible promoter and a3′ end region involved in transcription termination and polyadenylation;regenerating a population of transgenic plant lines from said transgenicplant cell; and identifying a stress tolerant plant line within thepopulation of transgenic plant lines.

The invention also provides DNA molecules comprising a plant-expressiblepromoter, operably linked to a DNA region, which when transcribed yieldsan ParG inhibitory RNA molecule, and to a 3′ end region involved intranscription termination and polyadenylation. The ParG inhibitory RNAmolecule may comprise a nucleotide sequence of at least 20 consecutivenucleotides of the nucleotide sequence of the ParG gene present in theplant cell (the endogenous ParG gene). The ParG inhibitory RNA moleculemay also comprise a nucleotide sequence of at least 20 consecutivenucleotides of the complement of the nucleotide sequence of the ParGgene present in the plant cell (the endogenous ParG gene). In yetanother embodiment, the parG inhibitory RNA may comprise a sense regioncomprising a nucleotide sequence of at least 20 consecutive nucleotidesof the nucleotide sequence of the ParG gene present in the plant celland an antisense region comprising a nucleotide sequence of at least 20consecutive nucleotides of the complement of the nucleotide sequence ofthe ParG gene present in the plant cell, wherein the sense and antisenseregion are capable of forming a double stranded RNA region comprisingsaid at least 20 consecutive nucleotides. The chimeric gene may furthercomprise a DNA region encoding a self-splicing ribozyme between said DNAregion coding for parG inhibitory RNA molecule and the 3′ end region.The chimeric gene may also comprise a nucleotide sequence of at least 21to 100 nucleotides of a nucleotide sequence encoding a proteincomprising the amino acid sequence of SEQ ID No 1, 2 or 16 or at least21 to 100 nucleotides of a nucleotide sequence of SEQ ID 3, 4, 15 or 23.

In yet another embodiment, the invention relates to plant cellcomprising the DNA molecule of the invention and plants consistingessentially of such plant cells, as well as to processes for producingstress tolerant plants, comprising the step of further crossing suchplants with another plant. Seeds and propagating material of such plantscomprising the chimeric genes of the invention are also provided.

The invention also relates to a method for obtaining stress tolerantplants comprising the steps of subjecting a plant cell line or a plantor plant line, to mutagenesis; identifying those plant cells or plantsthat have a mutation in an endogenous ParG gene; subjecting theidentified plant cells or plants to stress conditions and identifyingplant cells or plants that tolerate said stress conditions better thancontrol plants. Alternatively, plant cells or plants may be selected forresistance to ParG inhibitors and further treated as described in thisparagraph.

The invention further relates to a stress tolerant plant cell or planthaving a mutation in the endogenous ParG gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the poly-ADP ribosepolymeratization/depolymerization cycle by the action of PARP/PARG in aeukaryotic cell.

FIG. 2. Diagram of the NAD+ and ATP content of Arabidopsis lines underhigh light stress. Dark boxes represent NAD content under high lightconditions expressed as percentage of the value for NAD contentdetermined under low light conditions. Light boxes represent ATP contentunder high light conditions expressed as percentage of the value for ATPcontent determined under low light conditions.

FIG. 3. Diagram of the NAD+ and ATP content of corn lines under nitrogendepletion stress. Dark boxes represent NAD content while light boxesrepresent ATP content.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention is based, on the one hand, on the demonstration that cellsfrom stress resistant plant lines comprising a chimeric gene reducingthe PARP gene expression, exhibited a higher NAD/ATP content underadverse conditions than cells from untransformed plant lines. On theother hand, it has been observed that silencing of the expression ofPARG encoding gene in tobacco using a transient silencing RNA vectorbased on satellite viruses resulted in a similar phenotype as thatobserved for silencing of PARP encoding gene using the same silencingsystem. Furthermore, silencing the expression of PARG encoding gene inplants, such as Arabidopsis and tobacco, resulted in plants that weremore resistant to stress conditions, such as e.g. those imposed by highlight conditions.

Although not intending to limit the invention to a specific mode ofaction, it is expected that silencing of PARG gene expression results ina similar phenotype as silencing of PARP gene expression for thefollowing reasons. As can be seen from FIG. 1, polymerization of ADPribose catalyzed by PARP, consuming NAD, is followed by depolymerizationof poly ADP ribose, catalyzed by PARG. Poly ADP ribosylation of the PARPprotein itself results in inactivation of the PARP protein. The speed atwhich the ADP ribose polymerization/depolymerization cycle occurs inplant cells, leading to NAD depletion and consequently ATP depletion,can be slowed down or stopped by reduction of the PARP gene expressionor of the enzymatic activity of PARP. As a result, plant cells, andplants comprising such cells are more resistant to adverse conditions.The data provided here indicate that a similar effect can be obtainedthrough slowing down or stopping the cycle by reduction of the PARG geneexpression or PARG activity.

The invention relates to reduction of plant cell death in response toadverse environmental conditions, and consequently to enhanced stressresistance, by altering the level of expression of ParG genes, or byaltering the activity or the apparent activity of PARG proteins in thatplant cell. Conveniently, the level of expression of ParG genes may becontrolled genetically by introduction of chimeric genes altering theexpression of ParG genes, or by altering the endogenous PARG encodinggenes, including the expression signals.

In one embodiment of the invention, a method for producing plantstolerant to stress conditions or adverse growing conditions is providedcomprising the steps of:

providing plant cells with a chimeric gene to create transgenic plantcells, wherein the chimeric gene comprises the following operably linkedDNA fragments:

-   -   a plant-expressible promoter;    -   a DNA region, which when transcribed yields a ParG inhibitory        RNA molecule;    -   a 3′ end region involved in transcription termination and        polyadenylation;

regenerating a population of transgenic plant lines from said transgenicplant cell; and

identifying a stress tolerant plant line within said population oftransgenic plant lines.

As used herein “a stress tolerant plant” or “a plant tolerant to stressconditions or adverse growing conditions” is a plant (particularly aplant obtained according to the methods of the invention), which, whensubjected to adverse growing conditions for a period of time, such asbut not limited to drought, high temperatures, limited supply ofnutrients (particularly nitrogen), high light intensities, grows betterthan a control plant not treated according to the methods of theinvention. This will usually be apparent from the general appearance ofthe plants and may be measured e.g., by increased biomass production,continued vegetative growth under adverse conditions or higher seedyield. Stress tolerant plant have a broader growth spectrum, i.e. theyare able to withstand a broader range of climatological and otherabiotic changes, without yield penalty. Biochemically, stress tolerancemay be apparent as the higher NAD⁺-NADH/ATP content and lower productionof reactive oxygen species of stress tolerant plants compared to controlplants under stress condition. Stress tolerance may also be apparent asthe higher chlorophyll content, higher photosynthesis and lowerchlorophyll fluorescence under stress conditions in stress tolerantplants compared to control plants under the same conditions.

It will be clear that it is also not required that the plant be growncontinuously under the adverse conditions for the stress tolerance tobecome apparent. Usually, the difference in stress tolerance between aplant or plant cell according to the invention and a control plant orplant cell will become apparent even when only a relatively short periodof adverse conditions is encountered during growth.

As used herein, a “ParG inhibitory RNA molecule” is an RNA molecule thatis capable of decreasing the expression of the endogenous PARG encodinggenes of a plant cell, preferably through post-transcriptionalsilencing. It will be clear that even when a ParG inhibitory RNAmolecule decreases the expression of a PARG encoding gene throughpost-transcriptional silencing, such an RNA molecule may also exertother functions within a cell, such as e.g. guiding DNA methylation ofthe endogenous ParG gene, again ultimately leading to decreasedexpression of the PARG encoding gene. Also, expression of the endogenousPARG encoding genes of a plant cell may be reduced by transcriptionalsilencing, e.g., by using RNAi or dsRNA targeted against the promoterregion of the endogenous ParG gene.

As used herein, a “PARG encoding gene” or a “ParG gene” is a genecapable of encoding a PARG (poly ADP ribose glycohydrolase) protein,wherein the PARG protein catalyzes the depolymerization of polyADP-ribose, by releasing free ADP ribose units either by endoglycolyticor exoglycolytic action.

PARG encoding genes may comprise a nucleotide sequence encoding aprotein comprising the amino acid sequence of SEQ ID No 1 (Arabidopsisthaliana) or of SEQ ID No 2 (Solanum tuberosum) or of SEQ ID No 16(Oryza sativa) or parts thereof, such as a DNA fragment comprising thenucleotide sequence of SEQ ID No 3 or SEQ ID 4 or SEQ ID No 15. or SEQID 23 (Zea mays).

However, it will be clear that the skilled person can isolate variantDNA sequences from other plant species, by hybridization with a probederived from the above mentioned PARG encoding genes from plant species,or even with a probe derived from the above mentioned PARG encodinggenes from animal species. To this end, the probes should preferablyhave a nucleotide sequence comprising at least 40 consecutivenucleotides from the coding region of those mentioned PARG encodinggenes sequences, preferably from the coding region of SEQ ID No 3 or SEQID No 4. The probes may however comprise longer regions of nucleotidesequences derived from the ParG genes, such as about 50, 60, 75, 100,200 or 500 consecutive nucleotides from any of the mentioned ParG genes.Preferably, the probe should comprise a nucleotide sequence coding forone of the highly conserved regions of the catalytic domain, which havebeen identified by aligning the different PARG proteins from animals.These regions are also present in the identified PARG protein fromArabidopsis thaliana and comprise the amino acid sequence LXVDFANXXXGGG(corresponding to SEQ ID No 1 from the amino acid at position 252 to theamino acid at position 264; X may be any amino acid)LXVDFANXXXGGGXXXXGXVQEEIRF (corresponding to SEQ ID No 1 from the aminoacid at position 252 to the amino acid at position 277) orLXVDFANXXXGGGXXXXGXVQEEIRFXXXPE (corresponding to SEQ ID No 1 from theamino acid at position 252 to the amino acid at position 282),TGXWGCGXFXGD (corresponding to SEQ ID No 1 from the amino acid atposition 449 to the amino acid at position 460) or TGXWGCGAFXGDXXLKXXXQ(corresponding to SEQ ID No 1 from the amino acid at position 449 to theamino acid at position 468). Other conserved regions have the amino acidsequence DXXXRXXXXAIDA (corresponding to SEQ ID No 1 from the amino acidat position 335 to the amino acid at position 344) or REXXKAXXGF(corresponding to SEQ ID No 1 from the amino acid at position 360 to theamino acid at position 369) or GXXXXSXYTGY (corresponding to SEQ ID No 1from the amino acid at position 303 to the amino acid at position 313).Hybridization should preferably be under stringent conditions.

“Stringent hybridization conditions” as used herein mean thathybridization will generally occur if there is at least 95% andpreferably at least 97% sequence identity between the probe and thetarget sequence. Examples of stringent hybridization conditions areovernight incubation in a solution comprising 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared carrier DNA such as salmon sperm DNA, followed by washing thehybridization support in 0.1×SSC at approximately 65° C., e.g. for about10 min (twice). Other hybridization and wash conditions are well knownand are exemplified in Sambrook et al, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y. (1989), particularlychapter 11.

Alternatively, ParG encoding genes or parts thereof may also be isolatedby PCR based techniques, using as primers oligonucleotides comprising atleast 20 consecutive nucleotides from a nucleotide sequence of thementioned PARG encoding genes or the complement thereof. Such primersmay comprise a nucleotide sequence encoding a conserved region, asmentioned above, or be complementary to such a nucleotide sequence.Oligonucleotides which may be used for that purpose may comprise thenucleotide sequence of either or SEQ ID No. 5, SEQ ID No 6., SEQ ID No.7 or SEQ ID No. 8. Oligonucleotides which may be used may also bedegenerate, such as the oligonucleotide primers of SEQ ID No 17, SEQ IDNo 18, SEQ ID No 19; SEQ ID No 20, SEQ ID No 21 or SEQ ID No 22.

Specific PCR fragments from ParG genes may e.g., be obtained by usingcombinations of the oligonucleotides having the nucleotide sequence ofSEQ ID No. 5 and SEQ ID No 6 using e.g., Arabidopsis genomic DNA or cDNAas a template DNA, or by using combinations of the oligonucleotideshaving the nucleotide sequence of SEQ ID No. 7 and SEQ ID No 8 usinge.g., potato genomic DNA or cDNA as a template DNA, under stringentannealing conditions.

The isolated sequences may encode a functional PARG protein or a partthereof. Preferably the isolated sequences should comprise a nucleotidesequence coding for one or more of the highly conserved regions from thecatalytic domain of PARG proteins as mentioned elsewhere.

However, for the purpose of the invention is not required that theisolated sequences encode a functional ParG protein nor that a completecoding region is isolated. Indeed, all that is required for theinvention is that a chimeric gene can be designed or produced, based onthe identified or isolated sequence of the endogenous ParG gene from aplant, which is capable of producing a ParG inhibitory RNA. Severalalternative methods are available to produce such a ParG inhibitory RNAmolecule.

In one embodiment, the ParG inhibitory RNA molecule encoding chimericgene is based on the so-called antisense technology. In other words, thecoding region of the chimeric gene comprises a nucleotide sequence of atleast 20 consecutive nucleotides of the complement of the nucleotidesequence of the endogenous ParG gene of the plant cell or plant, theexpression of which is targeted to be reduced. Such a chimeric gene maybe conveniently constructed by operably linking a DNA fragmentcomprising at least 20 nucleotides from the isolated or identified ParGgene, or part of such a gene, in inverse orientation, to a plantexpressible promoter and 3′end formation region involved intranscription termination and polyadenylation. It will be immediatelyclear that there is no need to know the exact nucleotide sequence orcomplete nucleotide sequence of such a DNA fragment from an isolatedParG gene.

In another embodiment the ParG inhibitory RNA molecule encoding chimericgene is based on the so-called co-suppression technology. In otherwords, the coding region of the chimeric gene comprises a nucleotidesequence of at least 20 consecutive nucleotides of the nucleotidesequence of the endogenous ParG gene of the plant cell or plant, theexpression of which is targeted to be reduced. Such a chimeric gene maybe conveniently constructed by operably linking a DNA fragmentcomprising at least 20 nucleotides from the isolated or identified ParGgene, or part of such a gene, in direct orientation, to a plantexpressible promoter and 3′end formation region involved intranscription termination and polyadenylation. Again it is not requiredto know the exact nucleotide sequence of the used DNA fragment from theisolated ParG gene.

The efficiency of the above mentioned chimeric genes in reducing theexpression of the endogenous ParG gene may be further enhanced byinclusion of DNA elements which result in the expression of aberrant,unpolyadenylated ParG inhibitory RNA molecules. One such DNA elementsuitable for that purpose is a DNA region encoding a self-splicingribozyme, as described in WO 00/01133.

The efficiency or the above mentioned chimeric genes in reducing theexpression of the endogenous ParG gene of a plant cell may also befurther enhanced by including into one plant cell simultaneously achimeric gene as herein described encoding a antisense ParG inhibitoryRNA molecule and a chimeric gene as herein described encoding a senseParG inhibitory RNA molecule, wherein said antisense and sense ParGinhibitory RNA molecules are capable of forming a double stranded RNAregion by base pairing between the mentioned at least 20 consecutivenucleotides, as described in WO 99/53050.

As further described in WO 99/53050, the sense and antisense ParGinhibitory RNA regions, capable of forming a double stranded RNA regionmay be present in one RNA molecule, preferably separated by a spacerregion. The spacer region may comprise an intron sequence. Such achimeric gene may be conveniently constructed by operably linking a DNAfragment comprising at least 20 nucleotides from the isolated oridentified endogenous ParG gene, the expression of which is targeted tobe reduced, in an inverted repeat, to a plant expressible promoter and3′ end formation region involved in transcription termination andpolyadenylation. To achieve the construction of such a chimeric gene,use can be made of the vectors described in WO 02/059294

An embodiment of the invention thus concerns a method for obtaining astress tolerant plant line comprising the steps of

providing plant cells with a chimeric gene to create transgenic plantcells, wherein the chimeric gene comprises the following operably linkedDNA fragments:

-   -   a plant-expressible promoter,    -   a DNA region, which when transcribed yields a ParG inhibitory        RNA molecule comprising a nucleotide sequence of at least 20        consecutive nucleotides of the nucleotide sequence of the ParG        gene present in said plant cell; or    -   a DNA region, which when transcribed yields a ParG inhibitory        RNA molecule comprising a nucleotide sequence of at least 20        consecutive nucleotides of the complement of the nucleotide        sequence of the ParG gene present in said plant cell; or    -   a DNA region, which when transcribed yields a ParG inhibitory        RNA molecule comprising a sense region comprising a nucleotide        sequence of at least 20 consecutive nucleotides of the        nucleotide sequence of the ParG gene present in said plant cell        and an antisense region comprising a nucleotide sequence of at        least 20 consecutive nucleotides of the complement of the        nucleotide sequence of the ParG gene present in said plant cell,        wherein said sense and antisense region are capable of forming a        double stranded RNA region comprising said at least 20        consecutive nucleotides.    -   a 3′ end region involved in transcription termination and        polyadenylation;

regenerating a population of transgenic plant lines from said transgenicplant cell; and

identifying a stress tolerant plant line within said population oftransgenic plant lines.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA region which is functionallyor structurally defined, may comprise additional DNA regions etc.

It will thus be clear that the minimum nucleotide sequence of theantisense or sense RNA region of about 20 nt of the ParG coding regionmay be comprised within a larger RNA molecule, varying in size from 20nt to a length equal to the size of the target gene.

The mentioned antisense or sense nucleotide regions may thus be aboutfrom about 21 nt to about 5000 nt long, such as 21 nt, 40 nt, 50 nt, 100nt, 200 nt, 300 nt, 500 nt, 1000 nt, 2000 nt or even about 5000 nt orlarger in length.

Moreover, it is not required for the purpose of the invention that thenucleotide sequence of the used inhibitory ParG RNA molecule or theencoding region of the chimeric gene, is completely identical orcomplementary to the endogenous ParG gene the expression of which istargeted to be reduced in the plant cell. The longer the sequence, theless stringent the requirement for the overall sequence identity is.Thus, the sense or antisense regions may have an overall sequenceidentity of about 40% or 50% or 60% or 70% or 80% or 90% or 100% to thenucleotide sequence of the endogenous ParG gene or the complementthereof. However, as mentioned antisense or sense regions shouldcomprise a nucleotide sequence of 20 consecutive nucleotides havingabout 100% sequence identity to the nucleotide sequence of theendogenous ParG gene. Preferably the stretch of about 100% sequenceidentity should be about 50, 75 or 100 nt.

For the purpose of this invention, the “sequence identity” of tworelated nucleotide sequences, expressed as a percentage, refers to thenumber of positions in the two optimally aligned sequences which haveidentical residues (×100) divided by the number of positions compared. Agap, i.e. a position in an alignment where a residue is present in onesequence but not in the other is regarded as a position withnon-identical residues. The alignment of the two sequences is performedby the Needleman and Wunsch algorithm (Needleman and Wunsch 1970)Computer-assisted sequence alignment, can be conveniently performedusing standard software program such as GAP which is part of theWisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wis.,USA) using the default scoring matrix with a gap creation penalty of 50and a gap extension penalty of 3.

It will be clear that whenever nucleotide sequences of RNA molecules aredefined by reference to nucleotide sequence of corresponding DNAmolecules, the thymine (T) in the nucleotide sequence should be replacedby uracil (U). Whether reference is made to RNA or DNA molecules will beclear from the context of the application.

It will also be clear that chimeric genes capable of producinginhibitory ParG genes for a particular ParG gene in a particular plantvariety or plant species, may also be used to inhibit ParG geneexpression in other plant varieties or plant species. Indeed, whensufficient homology exists between the ParG inhibitory RNA region andthe ParG gene, or when the ParG genes share the same stretch of 19nucleotides, expression of those other genes will also bedown-regulated.

In view of the potential role of ParG in nucleic acid metabolism, it maybe advantageous that the expression of the endogenous ParG gene by theParG inhibitory RNA is not completely inhibited. Downregulating theexpression of a particular gene by gene silencing through theintroduction of a chimeric gene encoding ParG inhibitory RNA will resultin a population of different transgenic lines, exhibiting a distributionof different degrees of silencing of the ParG gene. The population willthus contain individual transgenic plant lines, wherein the endogenousParG gene is silenced to the required degree of silencing. A personskilled in the art can easily identify such plant lines, e.g. bysubjecting the plant lines to a particular adverse condition, such ahigh light intensity, oxidative stress, drought, heat etc. and selectingthose plants which perform satisfactory and survive best the treatment.

As used herein, the term “promoter” denotes any DNA which is recognizedand bound (directly or indirectly) by a DNA-dependent RNA-polymeraseduring initiation of transcription. A promoter includes thetranscription initiation site, and binding sites for transcriptioninitiation factors and RNA polymerase, and can comprise various othersites (e.g., enhancers), at which gene expression regulatory proteinsmay bind.

The term “regulatory region”, as used herein, means any DNA, that isinvolved in driving transcription and controlling (i.e., regulating) thetiming and level of transcription of a given DNA sequence, such as a DNAcoding for a protein or polypeptide. For example, a 5′ regulatory region(or “promoter region”) is a DNA sequence located upstream (i.e., 5′) ofa coding sequence and which comprises the promoter and the5′-untranslated leader sequence. A 3′ regulatory region is a DNAsequence located downstream (i.e., 3′) of the coding sequence and whichcomprises suitable transcription termination (and/or regulation)signals, including one or more polyadenylation signals.

In one embodiment of the invention the promoter is a constitutivepromoter. In another embodiment of the invention, the promoter activityis enhanced by external or internal stimuli (inducible promoter), suchas but not limited to hormones, chemical compounds, mechanical impulses,abiotic or biotic stress conditions. The activity of the promoter mayalso regulated in a temporal or spatial manner (tissue-specificpromoters; developmentally regulated promoters).

For the purpose of the invention, the promoter is a plant-expressiblepromoter. As used herein, the term “plant-expressible promoter” means aDNA sequence which is capable of controlling (initiating) transcriptionin a plant cell. This includes any promoter of plant origin, but alsoany promoter of non-plant origin which is capable of directingtranscription in a plant cell, i.e., certain promoters of viral orbacterial origin such as the CaMV35S (Hapster et al., 1988), thesubterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNAgene promoters but also tissue-specific or organ-specific promotersincluding but not limited to seed-specific promoters (e.g., WO89/03887),organ-primordia specific promoters (An et al., 1996), stem-specificpromoters (Keller et al., 1988), leaf specific promoters (Hudspeth etal., 1989), mesophyl-specific promoters (such as the light-inducibleRubisco promoters), root-specific promoters (Keller et al., 1989),tuber-specific promoters (Keil et al., 1989), vascular tissue specificpromoters (Peleman et al., 1989), stamen-selective promoters (WO89/10396, WO 92/13956), dehiscence zone specific promoters (WO 97/13865)and the like.

Methods for the introduction of chimeric genes into plants are wellknown in the art and include Agrobacterium-mediated transformation,particle gun delivery, microinjection, electroporation of intact cells,polyethyleneglycol-mediated protoplast transformation, electroporationof protoplasts, liposome-mediated transformation, silicon-whiskersmediated transformation etc. The transformed cells obtained in this waymay then be regenerated into mature fertile plants.

The transgenic plant cells and plant lines according to the inventionmay further comprise chimeric genes which will reduce the expression ofPARP genes as described in WO 00/04173. These further chimeric genes maybe introduced e.g. by crossing the transgenic plant lines of the currentinvention with transgenic plants containing PARP gene expressionreducing chimeric genes. Transgenic plant cells or plant lines may alsobe obtained by introducing or transforming the chimeric genes of theinvention into transgenic plant cells comprising the PARP geneexpression reducing chimeric genes or vice versa. Alternatively, thePARP and PARG inhibitory RNA regions may be encoded by one chimeric geneand transcribed as one RNA molecule.

The chimeric genes of the invention (or the inhibitory RNA moleculescorresponding thereto) may also be introduced into plant cells in atransient manner, e.g. using the viral vectors, such as viral RNAvectors as described in WO 00/63397 or WO 02/13964.

Having read this specification, it will be immediately clear to theskilled artisan, that mutant plant cells and plant lines, wherein thePARG activity is reduced may be used to the same effect as thetransgenic plant cells and plant lines described herein. Mutants in ParGgene of a plant cell or plant may be easily identified using screeningmethods known in the art, whereby chemical mutagenesis, such as e.g.,EMS mutagenesis, is combined with sensitive detection methods (such ase.g., denaturing HPLC). An example of such a technique is the so-called“Targeted Induced Local Lesions in Genomes” method as described inMcCallum et al, Plant Physiology 123 439-442 or WO 01/75167. However,other methods to detect mutations in particular genome regions or evenalleles, are also available and include screening of libraries ofexisting or newly generated insertion mutant plant lines, whereby poolsof genomic DNA of these mutant plant lines are subjected to PCRamplification using primers specific for the inserted DNA fragment andprimers specific for the genomic region or allele, wherein the insertionis expected (see e.g. Maes et al., 1999, Trends in Plant Science, 4, pp90-96).

Plant cell lines and plant lines may also be subjected to mutagenesis byselection for resistance to ParG inhibitors, such as gallotannines.(Ying, et al. (2001). Proc. Natl. Acad. Sci. USA 98(21), 12227-12232;Ying, W., Swanson, R. A. (2000). NeuroReport 11 (7), 1385-1388.

Thus, methods are available in the art to identify plant cells and plantlines comprising a mutation in the ParG gene. This population of mutantcells or plant lines can then be subjected to different abioticstresses, and their phenotype or survival can be easily determined.Additionally, the NAD and/or the ATP content of the stressed cells canbe determined and compared to results of such determinations ofunstressed cells. In stress tolerant cells, the reduction of NAD contentunder stress conditions should when compared with unstressed cells,should be lower than for corresponding control cells.

It is also an object of the invention to provide plant cells and plantscontaining the chimeric genes or the RNA molecules according to theinvention. Gametes, seeds, embryos, either zygotic or somatic, progenyor hybrids of plants comprising the chimeric genes of the presentinvention, which are produced by traditional breeding methods are alsoincluded within the scope of the present invention.

The plants obtained by the methods described herein may be furthercrossed by traditional breeding techniques with other plants to obtainstress tolerant progeny plants comprising the chimeric genes of thepresent invention.

The methods and means described herein are believed to be suitable forall plant cells and plants, both dicotyledonous and monocotyledonousplant cells and plants including but not limited to cotton, Brassicavegetables, oilseed rape, wheat, corn or maize, barley, alfalfa,peanuts, sunflowers, rice, oats, sugarcane, soybean, turf grasses,barley, rye, sorghum, sugar cane, vegetables (including chicory,lettuce, tomato, zucchini, bell pepper, eggplant, cucumber, melon,onion, leek), tobacco, potato, sugarbeet, papaya, pineapple, mango,Arabidopsis thaliana, but also plants used in horticulture, floricultureor forestry (poplar, fir, eucalyptus etc.).

The following non-limiting Examples describe method and means forincreasing stress tolerance in plants according to the invention.

Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA. Standard materials and methods for plant molecular workare described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK. Other references for standard molecularbiology techniques include Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, NY, Volumes I and II of Brown (1998) Molecular BiologyLabFax, Second Edition, Academic Press (UK). Standard materials andmethods for polymerase chain reactions can be found in Dieffenbach andDveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring HarborLaboratory Press, and in McPherson at al. (2000) PCR—Basics: FromBackground to Bench, First Edition, Springer Verlag, Germany.

Throughout the description and Examples, reference is made to thefollowing sequences:

SEQ ID No. 1: amino acid sequence of the ParG protein from Arabidopsisthaliana.

SEQ ID No. 2: amino acid sequence of part of the ParG protein fromSolanum tuberosum.

SEQ ID No. 3: nucleotide sequence encoding the ParG protein fromArabidopsis thaliana.

SEQ ID No. 4: nucleotide sequence encoding the part of the ParG proteinfrom Solanum tuberosum.

SEQ ID No. 5: nucleotide sequence of an oligonucleotide primer suitablefor PCR amplification of part of a ParG protein encoding DNA fragment.

SEQ ID No. 6: nucleotide sequence of an oligonucleotide primer suitablefor PCR amplification of part of a ParG protein encoding DNA fragment.

SEQ ID No. 7: nucleotide sequence of an oligonucleotide primer suitablefor PCR amplification of part of a ParG protein encoding DNA fragment.

SEQ ID No. 8: nucleotide sequence of an oligonucleotide primer suitablefor PCR amplification of part of a ParG protein encoding DNA fragment.

SEQ ID No. 9: nucleotide sequence of the T-DNA vector containing theParG expression reducing chimeric gene based on the Arabidopsis ParGgene sequence.

SEQ ID No. 10: amino acid sequence of conserved sequence 1 of PARGproteins.

SEQ ID No. 11: amino acid sequence of conserved sequence 2 of PARGproteins.

SEQ ID No. 12: amino acid sequence of conserved sequence 3 of PARGproteins.

SEQ ID No. 13: amino acid sequence of conserved sequence 4 of PARGproteins.

SEQ ID No. 14: amino acid sequence of conserved sequence 5 of PARGproteins.

SEQ ID No. 15: nucleotide sequence of the ParG protein from Oryzasativa.

SEQ ID No. 16: amino acid sequence of the ParG protein from Oryzasativa.

SEQ ID No. 17: nucleotide sequence of an oligonucleotide primer PG1suitable for PCR amplification of part of a ParG protein encoding DNAfragment.

SEQ ID No. 18: nucleotide sequence of an oligonucleotide primer PG2suitable for PCR amplification of part of a ParG protein encoding DNAfragment.

SEQ ID No. 19: nucleotide sequence of an oligonucleotide primer PG3suitable for PCR amplification of part of a ParG protein encoding DNAfragment.

SEQ ID No. 20: nucleotide sequence of an oligonucleotide primer PG4suitable for PCR amplification of part of a ParG protein encoding DNAfragment.

SEQ ID No. 21: nucleotide sequence of an oligonucleotide primer PG5suitable for PCR amplification of part of a ParG protein encoding DNAfragment.

SEQ ID No. 22: nucleotide sequence of an oligonucleotide primer PG6suitable for PCR amplification of part of a ParG protein encoding DNAfragment.

SEQ ID No. 23: nucleotide sequence encoding a ParG protein from Zeamays.

SEQ ID No. 24: nucleotide sequence of a T-DNA vector comprising achimeric gene capable of reducing PARG expression

SEQ ID No. 25: nucleotide sequence of a T-DNA vector comprising achimeric gene capable of reducing PARG expression

EXAMPLES Example 1 Analysis of the Influence of Stress on EnergyProduction Efficiency of Transgenic Stress Tolerant Plant LinesContaining PARP Gene Expresssion Reducing Chimeric Genes

Hypocotyls of transgenic Brassica napus plants comprising PARP geneexpression reducing chimeric genes as described in WO 00/04173 werecultivated for 5 days on a growth medium. Explants were then transferredto liquid medium comprising 30 mg/L aspirin or acetylsalicylic acid(resulting in oxidative stress conditions) for one day. In controlexperiments, hypocotyls of non-transgenic Brassica napus plants N90-740were cultivated on the same growth medium and then incubated for one dayin liquid medium comprising 30 mg/L aspirin. In addition, hypocotyls ofboth the transgenic lines and the control line were cultivated on thesame growth medium without aspirin.

After the cultivation period, the ATP content of 125 explants wasdetermined for each experiment. Additionally, the oxygen consumed in 3hours by 125 explants was determined. The results are summarized inTable 1. The standard error of the mean was less than 6%. Whereas, theratio of moles ATP per mg consumed oxygen in the control plantsdecreased in the control plants when oxidative stress was applied, thesame ratio in the stress tolerant transgenic plant lines actuallyincreased under stress conditions, and was considerably higher (about24%) than in the control plants. The stress resistant transgenic linesthus maintained an constant energy production efficiency, whereas thecontrol lines exhibited an decreased energy production efficiency. Inaddition, superoxide production, expressed as a percentage of superoxideproduction in control plants not subjected to the oxidative stress, didnot increase in stress tolerant plants subjected to stress conditions.TABLE 1 Influence of stress on energy production efficiency of 5 dayscultured Brassica napus hypocotyl explants. O₂ moles mg/L ATP molesconsumed mg ATP in 3 hrs con- per 125 by 125 sumed Superoxide Plant lineStress explants explants O₂ production N90-740 None 12.4 × 2.96 4.19 ×100% (control) 10⁻⁷ 10⁻⁷ 30 mg/L 13.2 × 4.06 3.25 × 167% aspirin 10⁻⁷10⁻⁷ Transgenic None  9.3 × 2.33 3.99 × 108% line 10⁻⁷ 10⁻⁷ 30 mg/L 11.4× 2.82 4.04 × 100% aspirin 10⁻⁷ 10⁻⁷

In another experiment, the NAD+ and ATP content of 4 differenttransgenic Arabidopsis lines comprising PARP gene expression reducingchimeric genes as described in WO 00/04173 were determined under highand low light conditions, and compared to the values obtained for a nontransformed control line under the same conditions. The 4 differentlines exhibited different degrees of stress resistance as exhibited e.g.by their ability to withstand heat and/or drought conditions. The valuesobtained for the NAD and ATP contents under high light stress areexpressed as a percentage of the values for the NAD and ATP contentsunder low light conditions, and are plotted in FIG. 2.

The results show that high light stress leads to a significant NADreduction in control plant cells and in the transgenic plant line whichis the least stress resistant. The more stress resistant the transgenicplant lines are, the less signicifant the NAD reduction is under highlight stress conditions.

In another experiment, the NAD+ and ATP content of a segregatingpopulation resulting from a cross between transgenic corn linescomprising PARP gene expression reducing chimeric genes as described inWO 00/04173 and an untransformed corn line, were determined underconditions of nutrient (nitrogen) depletion, and compared to the valuesobtained for a non transformed control line under the same conditions.FIG. 3 is a graphic representation of the of the obtained results.Hemizygous and azygous lines were discriminated by verification for thepresence of the selectable marker gene. The NAD and ATP content wassignificantly higher in the hemizygous, stress tolerant plants than inthe untransformed control plants or the azygous plants.

Example 2 Construction of ParG Gene Expression Reducing Chimeric Genes

To reduce the expression of the PARG gene e.g. in Arabidopsis andrelated plants, a chimeric gene was constructed which is capableexpressing a dsRNA comprising both a sense and antisense region whichcan form a double stranded RNA. Such dsRNA is very effective in reducingthe expression of the genes with which is shares sequence homology, bypost-transcriptional silencing. The chimeric gene comprises thefollowing DNA fragments:

-   -   A promoter region from Cauliflower mosaic Virus (CaMV 35S);    -   A DNA fragment comprising 163 bp from the ParG gene from        Arabidopsis thaliana in direct orientation (Genbank Accession        number AF394690 from nucleotide position 973 to 1135);    -   A DNA fragment encoding intron 2 from the pdk gene from        Flaveria;    -   The DNA fragment comprising 163 bp from the ParG gene from        Arabidopsis thaliana in inverted orientation (Genbank Accession        number AF394690 from nucleotide position 973 to 1135)    -   A fragment of the 3′ untranslated end from the octopine        synthetase gene from Agrobacterium tumefaciens.

This chimeric gene was introduced in a T-DNA vector, between the leftand right border sequences from the T-DNA, together with a selectablemarker gene providing resistance to the herbicide phosphinotricin.

To reduce the expression of the PARG gene e.g. in potatoes and relatedplants, a chimeric gene is constructed which is capable expressing adsRNA comprising both a sense and antisense region of a cDNA sequencefrom potato, that is capable of encoding a protein having high sequenceidentity with the N-terminal part of the Arabidopsis PARG protein. Thechimeric gene comprises the following DNA fragments:

-   -   A promoter region from Cauliflower mosaic Virus (CaMV 35S);    -   A DNA fragment comprising a sequence of at least 100 bp from        ParG homologue from Solanum tuberosum in direct orientation        (Genbank Accession number BE340510);    -   A DNA fragment encoding intron 2 from the pdk gene from        Flaveria;    -   The DNA fragment comprising the sequence of at least 100 bp from        ParG homologue from Solanum tuberosum in inverted orientation        (Genbank Accession number BE340510);    -   A fragment of the 3′ untranslated end from the octopine        synthetase gene from Agrobacterium tumefaciens

This chimeric gene is introduced in a T-DNA vector, between the left andright border sequences from the T-DNA, together with a selectable markergene providing resistance to the herbicide phosphinotricin.

Example 3 Analysis of Transgenic Plant Lines Comprising ParG GeneExpression Reducing Chimeric Genes

The chimeric genes of Example 2 are introduced into Arabidopsis orpotato respectively, by Agrobacterium mediated transformation.

The population of obtained transgenic lines is subjected to thefollowing stress conditions, together with control plants:

-   -   Increased heat for a period of days (greenhouse) or hours (in        vitro)    -   Drought for a period of days    -   High light conditions for a period of days    -   Nutrient depletion

Individual plant lines surviving well the above mendioned stressconditions are selected.

The NAD content and ATP content for the above mentioned plants isdetermined under control and stress conditions.

Example 4 Quantitative Determination of NAD, ATP and Superoxide Radicalsin Plant Cells

Quantification of ATP in plant tissues was done basically as decribed byRawyler et al. (1999), Plant Physiol. 120, 293-300. The assay was usedfor the determination of the ATP content of hypocotyl explants that werecultured for 4-5 days on A2S3 medium or 2 weeks old in vitro culturedArabidopsis plants. All manipulations are performed on crushed iceunless otherwhise indicated.

ATP Extraction

-   -   Freeze plant material with liquid nitrogen        -   100 hypocotyl explants        -   ±700 mg Arabidopsis plants (roots+shoots) (about 32-37            18-days old C24 plants)    -   Put frozen hypocotyls in mortar and add 6 ml of 6% perchloric        acid.    -   Extraction can be done at room temperature using a pestle. After        extraction, put samples as soon as possible on ice.    -   Centrifuge at 24,000 g (Sorvall, SS34 rotor at 14,000 rpm) for        10 min. at 4° C.    -   The supernatant is neutralized with 5M K₂CO₃ (add 350 μl of 5M        K₂CO₃ to 3 ml of supernatant).    -   KClO₄ is removed by spinning as described above.

Quantitative bioluminescent determination of ATP

-   -   The ATP bioluminescent assay kit from Sigma is used (FL-M).    -   Dilute extract 6000×(about 6 mL extract from which 100 μl is        taken, that is diluted 1000 times) The dilutions are made with        the ‘ATP assay mix dilution buffer’ (FL-AAB) of the ATP        bioluminescent assay kit    -   The amount of light that is produced is measured with the        TD-20/20 luminometer of Turner Designs (Sunnyvale, USA).    -   Standard curve: disolve ATP standard of kit (FL-MS) in 10 ml of        water (2×10⁻⁶ moles)

Quantification of NAD+ and NADH in plant tissues was performed,essentially as described by Karp et al. (1983) or Filipovic et al.(1999) on the following plant material:

-   -   Brassica napus: 150 5-days cultured hypocotyl explants/sample        Arabidopsis: 1000 mg 18-days old in vitro grown plants        (shoots+roots)/sample (corresponds to ±60 C24 plants)

Assay Solution

-   -   (A) For measuring NADH:        -   25 mM potassium phosphate buffer pH7        -   0.1 mM DTT        -   3 μM FMN (Fluka, 83810)        -   30 μM n-decanal (Sigma, D-7384)    -   (B) For measuring NAD⁺+NADH:        -   idem as for measuring NADH alone+2 μg/mL alcohol            dehydrogenase (Roche, 102 717)

Extraction

-   -   Freeze with liquid nitrogen    -   Put frozen plant material in cooled mortar (cooled at −20° C.)        and add 5 mL extraction buffer    -   Grind material using a pestle    -   Centrifuge at 24 000 g (Sorvall, SS34 rotor at 14 000 rpm) for        15 minutes at 4° C.    -   Take 1 mL of supernatant for analysis

Assay

NADH

-   -   390 μL of assay solution A    -   +10 μL extract    -   +2 μL NAD(P)H:FMN oxidoreductase    -   +100 μL luciferase solution        NAD⁺+NADH    -   390 μL of assay solution B    -   +10 μL extract    -   2 minutes at room temperature    -   +2 μL NAD(P)H:FMN oxidoreductase    -   +100 μL luciferase solution        The amount of light that is produced is measured with the        TD-20/20 luminometer of Turner Designs (Sunnyvale, USA)        NADH-Standard

NADH stock solution: 1 mM (7.1 mg/10 mL H₂O)

-   -   NADH: disodium salt, Roche, 107 735    -   Dilution series in 10 mM potassium phosphate buffer pH7: (10⁻²);        5×10⁻³;    -   2×10⁻³; 10⁻³; 5×10⁻⁴    -   Add 10 μL of dilutions in 390 μL of assay solution A and perform        reaction    -   Make standard curve

Superoxide radicals production was measured by quantifying the reductionof XTT as described in De Block and De Brouwer (2002) Plant Physiol.Biochem. 40, 845-852

Brassica Napus

Media and Reaction Buffers

Sowing Medium (Medium 201):

-   -   Half concentrated Murashige and Skoog salts    -   2% sucrose    -   pH 5.8    -   0.6% agar (Difco Bacto Agar)    -   250 mg/l triacillin        Callus Inducing Medium A2S3:    -   MS medium, 0.5 g/l Mes (pH 5.8), 3% sucrose, 40 mg/ adenine-SO₄,    -   0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/l NAA, 1 mg/l BAP, 250 mg/l        triacillin        Incubation Medium:    -   25 mM K-phosphate buffer pH5.8    -   2% sucrose    -   1 drop Tween20 for 25 ml medium        Reaction Buffer    -   50 mM K-phosphate buffer pH7.4    -   1 mM sodium,        3′-{1-[phenylamino-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)=XTT        (bts, Germany, cat no. 2525) 1 drop Tween20 for 25 ml buffer

Sterilization of seeds—pregermination of seeds—growing of the seedlings.Seeds are soaked in 70% ethanol for 2 min, then surface-sterilized for15 min in a sodium hypochlorite solution (with about 6% active chlorine)containing 0.1% Tween20. Finally, the seeds are rinsed with 1l ofsterile tap water. Incubate seeds for at least one hour in sterile tapwater (to allow diffusion from seeds of components that may inhibitgermination). Seeds are put in 250 ml erlenmeyer flasks containing 50 mlof sterile tap water (+250 mg/l triacillin). Shake for about 20 hours.Seeds from which the radicle is protruded are put in Vitro Ventcontainers from Duchefa containing about 125 ml of sowing medium (10seeds/vessel, not too many to reduce loss of seed by contamination). Theseeds are germinated at ±94° C. and 10-30:Einstein/s⁻¹ m⁻² with adaylength of 16 h.

Preculture of the hypocotyl explants and induction of stress

-   -   12-14 days after sowing, the hypocotyls are cut in about 7-10 mm        segments.    -   The hypocotyl explants (25 hypocotyls/Optilux Petridish, Falcon        S1005, Denmark) are cultured for 5 days on medium A2S3 at 25° C.        (at 10-30□ Einstein/s⁻¹m⁻²).

XTT-Assay

-   -   Transfer 150 hypocotyl explants to a 50 ml Falcon tube.    -   Wash with reaction buffer (without XTT).    -   Add 20 mL reaction buffer+XTT.        -   (explants have to be submerged, but do not vacuum            infiltrate)    -   Incubate in the dark at 26° C. for about 3 hours    -   Measure the absorption of the reaction medium at 470 nm

Arabidopsis Thaliana

Media and reaction buffers

Plant Medium:

-   -   Half concentrated Murashige and Skoog salts    -   B5 vitamins    -   1.5% sucrose    -   pH 5.8    -   0.7% Difco agar        Incubation Medium:    -   10 mM K-phosphate buffer pH5.8    -   2% sucrose    -   1 drop Tween20 for 25 ml medium        Reaction Buffer:    -   50 mM K-phosphate buffer pH7.4    -   1 mM sodium,        3′-{1-[phenylamino-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)=XTT        (bts, Germany, cat no. 2525)    -   1 drop Tween20 for 25 ml buffer

Arabidopsis Plants

-   -   Arabidopsis lines:        -   control        -   lines to test    -   Sterilization of Arabidopsis seeds:        -   2 min. 70% ethanol        -   10 min. bleach (6% active chlorine)+1 drop Tween 20 for 20            ml        -   solution        -   wash 5 times with sterile tap water    -   Pregermination of seeds:        -   In 9 cm Optilux Petridishes (Falcon) containing 12 ml            sterile tap water.    -   Low light overnight to 24 hours.    -   Growing of Arabidopsis plants        -   Seeds are sown in Intergrid Tissue Culture disks of Falcon            (nr. 3025) containing±125 ml of plant medium: 1 seed/grid.        -   Plants are grown at 24° C.            -   30μEinstein s⁻¹m⁻²        -   16 hours light-8 hours dark    -   for about 3 weeks (before bolting)

XTT-Assay

Control Condition (No Stress)

-   -   Harvest shoots (roots included) from agar plates and put them        directly in a 50 ml Falcon tube containing reaction buffer        (without XTT)        Stressed Shoots    -   Transfer shoots to 50 ml Falcon tubes containing reaction buffer        (without XTT)    -   Replace reaction buffer with buffer containing XTT (40 mL/tube)    -   Shoots have to be submerged, but do not vacuum infiltrate    -   Incubate in the dark at 26° C. for about 3 hours    -   Measure the absorption of the reaction medium at 470 nm

Quantification of respiration by measuring oxygen consumption using aClark polarographic electrode was done in the following way:

Plant Material

Brassica napus

-   -   150-200* hypocotyl explants Cultured for 5 days at 25° C.    -   (cfr. protocol vigour assay)    -   * 150 explants error<10%; 200 explants error<6%

Arabidopsis

-   -   For C24±1000 mg* in vitro plants (shoots+roots) (corresponds        with    -   ˜50 18-days old plants)    -   Pregerminate seeds before sowing    -   Grow for 18 days at 24° C.    -   (cfr. protocol in vitro growth Arabidopis)    -   * for error<8%

Incubation Media

Brassica napus

-   -   25 mM K-phosphate buffer pH5.8    -   2% sucrose    -   Tween20 (1 drop/25 ml)        Arabidopsis    -   10 mM K-phosphate buffer pH5.8    -   2% sucrose    -   Tween20 (1 drop/25 ml)        Before use, aerate (saturate with oxygen) medium well by        stirring for at least a few hours

Assay

-   -   Put explants in 100 ml glass bottle (Schott, Germany) filled        with incubation medium. Put the same weight of shoots in each        bottle (+700 mg)    -   Fill bottle to overflowing and close tightly (avoid large air        bubbles)    -   Fill also a bottle with incubation medium that does not contain        explants (blanco)    -   Incubate at 24° C. at low light for:        -   34 hours (Brassica napus)        -   3 hours (Arabidopsis)    -   Shake gently during incubation (to avoid oxygen depletion of        medium around explants)    -   Measure oxygen concentration (mg/l) of incubation media using an        hand-held dissolved oxygen meter (Cyberscan DO 310; Eutech        Instruments, Singapore)    -   mg/l consumed oxygen=[oxygen]blanco−[oxygen]sample.

Example 5 Analysis of Transgenic Plant Lines Comprising ParG GeneExpression Reducing Chimeric Genes

The chimeric genes of Example 2 were introduced into Arabidopsis anNicotiana tabacum c.v. Petit Havana SR1 by Agrobacterium mediatedtransformation.

Transgenic seeds were germinated on a medium containing MS salts/2; B5vitamins; 1.5% sucrose; pH5.8 and 0.7% Difco agar. Germinated seeds weresubject to low light (photosynthetic photon flux of about 30 μmol m⁻¹s⁻¹ for 14 to 18 days, after which the light intensity was increasedabout 6-fold (photosynthetic photon flux of about 190 μmol m⁻¹ s⁻¹).After 1 day, the NAD and NADH contents were determined using theenzymatic cycling method (Karp et al. (1983) Anal. Biochem. 128, pp175-180). A portion of the seedlings were cultivated further under highlight conditions for about 3 to about days, after which the damage wasscored. Damage was visible as darkening of the young leaves and shoottip, bleaching of older leaves and growth retardation. The results aresummarized in Table 1 for Arabidopsis and in Table 2 for tobacco. TABLE1 Analysis of Arabidopsis (Columbia). % NAD + NADH TTC-reducing Highlight content in 1 gram capacity vs tolerance of tissue (10⁻³ μM)control Non-transgenic control S 17.3 100 Transgenic line 9 R 28.2 NDTransgenic line 10 R 31.7 ND Transgenic line 11 ±R   26.5 ND Transgenicline 12 S 19.4 ND Transgenic line 26 R 33.2  55 Transgenic line 27 S21.3 100 Transgenic line 28 ±R   26.5  75 Transgenic line 29 S 17.7 102Transgenic line 30 R 28.3  66±R indicates that some dark pigmentation was observed.ND: not determined

TABLE 2 Analysis of Nicotiana tabacum c.v. Petit Havana SR1. %TTC-reducing High light capacity vs tolerance control Non-transgeniccontrol S 100  Transgenic line 1 R/S 88 Transgenic line 2 ±R   79Transgenic line 3 R 53±R indicates that some dark pigmentation was observed.R/S indicates tha the resistance phenotype was not very clear.

There is a positive correlation between the resistance to high lightstress in the transgenic plants and the NAD+NADH content of the cells.An inverse correlation can be seen between TTC reducing capacity andhigh light tolerance.

Example 6 Construction of ParG Gene Expression Reducing Chimeric GenesSuited for Use in Cereal Plants

To reduce the expression of the PARG gene e.g. in cereals such as riceor corn (maize) and related plants, a chimeric gene is constructed whichis capable expressing a dsRNA comprising both a sense and antisenseregion of nucleotide sequence from rice, that is capable of encoding aprotein having high sequence identity with PARG protein encodingnucleotide sequences. The chimeric gene comprises the following DNAfragments:

-   -   A promoter region from Cauliflower mosaic Virus (CaMV 35S);    -   A DNA fragment comprising a sequence of at least 100 bp from        ParG homologue from Oryza saliva (SEQ ID No 15) in direct        orientation;    -   A DNA fragment encoding intron 2 from the pdk gene from        Flaveria;    -   A DNA fragment comprising a sequence of at least 100 bp from        ParG homologue from Oryza sativa (SEQ ID No 15) in inverted        orientation;    -   A fragment of the 3′ untranslated end from the octopine        synthetase gene from Agrobacterium tumefaciens.

This chimeric gene is introduced in a T-DNA vector, between the left andright border sequences from the T-DNA, together with a selectable markergene providing resistance to e.g. the herbicide phosphinotricin.

To reduce the expression of the PARG gene e.g. in cereals such as riceor corn (maize) and related plants, a chimeric gene is constructed whichis capable expressing a dsRNA comprising both a sense and antisenseregion of nucleotide sequence from rice, that is capable of encoding aprotein having high sequence identity with PARG protein encodingnucleotide sequences. The chimeric gene comprises the following DNAfragments:

-   -   A promoter region from Cauliflower mosaic Virus (CaMV 35S);    -   A DNA fragment comprising a sequence of at least 100 bp from        ParG homologue from Zea mays (SEQ ID No 23) in direct        orientation;    -   A DNA fragment encoding intron 2 from the pdk gene from        Flaveria;    -   A DNA fragment comprising a sequence of at least 100 bp from        ParG homologue from Zea mays (SEQ ID No 23) in inverted        orientation;    -   A fragment of the 3′ untranslated end from the octopine        synthetase gene from Agrobacterium tumefaciens.

This chimeric gene is introduced in a T-DNA vector, between the left andright border sequences from the T-DNA, together with a selectable markergene providing resistance to e.g. the herbicide phosphinotricin. Thenucleotide sequence of two examples of such T-DNA vectors comprising twodifferent chimeric gences as described in the previous paragraph isrepresented in SEQ ID Nos 24 and 25.

Example 7 Analysis of Transgenic Plant Lines Comprising ParG GeneExpression Reducing Chimeric Genes

The chimeric genes of Example 6 are introduced into rice or cornrespectively, by Agrobacterium mediated transformation.

The population of obtained transgenic lines is subjected to thefollowing stress conditions, together with control plants:

-   -   Increased heat for a period of days (greenhouse) or hours (in        vitro)    -   Drought for a period of days    -   High light conditions for a period of days    -   Nutrient depletion

Individual plant lines surviving well the above mentioned stressconditions, or at least one thereof, are selected.

The NAD content and ATP content for the above mentioned plants isdetermined under control and stress conditions.

1. A method of producing a plant tolerant to stress conditionscomprising the steps of (a) providing plant cells with a chimeric geneto create transgenic plant cells, said chimeric gene comprising thefollowing operably linked DNA fragments (i) a plant-expressiblepromoter; (ii) a DNA region, which when transcribed yields an ParGinhibitory RNA molecule; (iii) a 3′ end region involved in transcriptiontermination and polyadenylation; (b) regenerating a population oftransgenic plant lines from said transgenic plant cell; and (c)identifying a stress tolerant plant line within said population oftransgenic plant lines.
 2. The method according to claim 1, wherein saidparG inhibitory RNA molecule comprises a nucleotide sequence of at least20 consecutive nucleotides of the nucleotide sequence of the ParG genepresent in said plant cell.
 3. The method according to claim 1, whereinsaid parG inhibitory RNA molecule comprises a nucleotide sequence of atleast 20 consecutive nucleotides of the complement of the nucleotidesequence of the ParG gene present in said plant cell.
 4. The methodaccording to claim 2 or 3, wherein said chimeric gene further comprisesa DNA region encoding a self-splicing ribozyme between said DNA regioncoding for said parG inhibitory RNA molecule and said 3′ end region. 5.The method according to claim 1, wherein said parG inhibitory RNAcomprises a sense region comprising a nucleotide sequence of at least 20consecutive nucleotides of the nucleotide sequence of the ParG genepresent in said plant cell and an antisense region comprising anucleotide sequence of at least 20 consecutive nucleotides of thecomplement of the nucleotide sequence of the ParG gene present in saidplant cell, wherein said sense and antisense region are capable offorming a double stranded RNA region comprising said at least 20consecutive nucleotides.
 6. The method according to claim 1, whereinsaid stress conditions are heat, drought, nutrient depletion, oxidativestress or high light conditions.
 7. The method according to claim 1,comprising further crossing said transgenic plant line with anotherplant line to obtain stress tolerant progeny plants.
 8. A method ofproducing a plant tolerant to stress conditions comprising the steps of:(a) isolating a DNA fragment of at least 100 bp comprising a part of theparG encoding gene of said plant; (b) producing a chimeric gene byoperably linking the following DNA fragments; (i) a plant expressiblepromoter region; (ii) said isolated DNA fragment comprising part of theparG encoding gene of said plant in direct orientation compared to thepromoter region; (iii) said isolated DNA fragment comprising part of theparG encoding gene of said plant in inverted orientation compared to thepromoter region; (iv) a 3′ end region involved in transcriptiontermination and polyadenylation; (c) providing plant cells with saidchimeric gene to create transgenic plant cells (d) regenerating apopulation of transgenic plant lines from said transgenic plant cell;and (e) identifying a stress tolerant plant line within said populationof transgenic plant lines.
 9. A DNA molecule comprising (i) aplant-expressible promoter; (ii) a DNA region, which when transcribedyields a ParG inhibitory RNA molecule; and (iii) a 3′ end regioninvolved in transcription termination and polyadenylation.
 10. The DNAmolecule according to claim 9, wherein said DNA region comprises anucleotide sequence of at least 21 to 100 nucleotides of a nucleotidesequence encoding a protein comprising the amino acid sequence of SEQ IDNo 1, 2 or 16 or at least 21 to 100 nucleotides of a nucleotide sequenceof SEQ ID 3, 4, 15 or
 23. 11. A plant cell comprising the DNA moleculeof claim 9 or
 10. 12. A plant consisting essentially of the plant cellsof claim
 11. 13. A process for producing stress tolerant plants,comprising the step of crossing a plant of claim 12 with another plant.14. Seeds and propagating material of a plant according to claim
 12. 15.Plants obtainable or obtained by the process of claim
 8. 16. A method ofproducing a plant tolerant to stress conditions comprising the steps of(a) providing plant cells with a chimeric gene to create transgenicplant cells, said chimeric gene comprising the following operably linkedDNA fragments (i) a plant-expressible promoter; (ii) a DNA region, whichwhen transcribed yields an ParG inhibitory RNA molecule, said DNA regioncomprising a nucleotide sequence of at least 21 to 100 nucleotides of anucleotide sequence encoding a protein comprising the amino acidsequence of SEQ ID No 1, 2 or 16 or at least 21 to 100 nucleotides of anucleotide sequence of SEQ ID 3, 4, 15 or 23; (iii) a 3′ end regioninvolved in transcription termination and polyadenylation; (b)regenerating a population of transgenic plant lines from said transgenicplant cell; and (c) identifying a stress tolerant plant line within saidpopulation of transgenic plant lines.
 17. A method of producing a planttolerant to stress conditions comprising the steps of (a) subjecting aplant cell line or a plant or plant line, to mutagenesis; (b)identifying those plant cells or plants that have a mutation in anendogenous ParG gene; (c) subjecting the identified plant cells orplants to stress conditions; (d) identifying plant cells or plants thattolerate said stress conditions better than control plants.
 18. A methodof producing a plant tolerant to stress conditions comprising the stepsof (a) selecting a plant cell line or a plant or plant line which isresistant to a ParG inhibitor; (b) identifying those plant cells orplants that have a mutation in an endogenous ParG gene; (c) subjectingthe identified plant cells or plants to stress conditions; (d)identifying plant cells or plants that tolerate said stress conditionsbetter than control plants.
 19. A stress tolerant plant cell or plantcomprising a mutation in an endogenous ParG gene.